Non-productive DNA damage binding by DNA glycosylase-like protein Mag2 from Schizosaccharomyces pombe

Abstract

Schizosaccharomyces pombe contains two paralogous proteins, Mag1 and Mag2, related to the helix-hairpin-helix (HhH) superfamily of alkylpurine DNA glycosylases from yeast and bacteria. Phylogenetic analysis of related proteins from four Schizosaccharomyces and other fungal species shows that the Mag1/Mag2 duplication is unique to the genus Schizosaccharomyces and most likely occurred in its ancestor. Mag1 excises N3- and N7-alkylguanines and 1,N(6)-ethenoadenine from DNA, whereas Mag2 has been reported to have no detectible alkylpurine base excision activity despite high sequence and active site similarity to Mag1. To understand this discrepancy we determined the crystal structure of Mag2 bound to abasic DNA and compared it to our previously determined Mag1-DNA structure. In contrast to Mag1, Mag2 does not flip the abasic moiety into the active site or stabilize the DNA strand 5' to the lesion, suggesting that it is incapable of forming a catalytically competent protein-DNA complex. Subtle differences in Mag1 and Mag2 interactions with the DNA duplex illustrate how Mag2 can stall at damage sites without fully engaging the lesion. We tested our structural predictions by mutational analysis of base excision and found a single amino acid responsible at least in part for Mag2's lack of activity. Substitution of Mag2 Asp56, which caps the helix at the base of the DNA intercalation loop, with the corresponding serine residue in Mag1 endows Mag2 with ɛA excision activity comparable to Mag1. This work provides novel insight into the chemical and physical determinants by which the HhH glycosylases engage DNA in a catalytically productive manner.

Figure 1. Phylogenetic history of the Mag family of proteins from Schizosaccharomyces and representative fungal relatives

Numbers above branches represent bootstrap support and branch lengths represent protein evolutionary rate in units of amino acid substitutions per site. The Mag phylogeny shows that the duplication of Mag1 and Mag2 is specific and unique to the Schizosaccharomyces clade and that both paralogs have been retained in all 4 Schizosaccharomyces species.

Figure 2. Crystal structure of S. pombe Mag2 bound to abasic-DNA

(A,B) Mag2 (blue ribbons, white solvent accessible surface) in complex with DNA (gold) containing a THF abasic site analog (green). The disordered stretch of DNA is colored salmon. (B) Schematic of Mag2-DNA interactions, colored as in panel A with HhH residues in cyan. Dotted lines represent either hydrogen bonds or van der Waals interactions. mc, main chain. (C) Superposition of Mag2/THF-DNA (colored as in panel A) onto S. pombe Mag1/THF-DNA (PDB ID 3S6I, silver). (D) Structure based sequence alignment of Mag1 and Mag2, generated with Swiss PDB Viewer [51]. Secondary structure elements are shown above the alignment. The HhH loop is represented as light blue cylinders. Nucleobase binding pocket residues are highlighted orange, intercalating plug and wedge residues are green, and the catalytic aspartate found in HhH glycosylases is yellow. Mag2 Asp56, responsible for diminished base excision activity, is highlighted magenta.

Figure 3. Mag2 does not flip abasic sites

(A) Stereoview of the region around the THF abasic site in Mag2. Protein and DNA are colored by carbon atom as in Figure 2. (B) Superposition of DNA bound to Mag1 (grey) and Mag2 (gold/green/salmon). The THF moiety is flipped out of the duplex in the Mag1 structure (denoted with an asterisk) but remains stacked in the duplex in the Mag2 structure (green). (C) Average B-factor per nucleotide between Mag2 (gold/green/salmon) and Mag1 (grey) complexes. The THF-containing and unmodified strands are shown as squares and circles respectively.

Figure 4. Mag2 has a capped helix dipole that inhibits base excision activity

(A) Close-up view of the DNA interrogating loop. The arrow denotes the direction of the dipole of helix D, with the positive end toward the DNA. The position of Asp56 is marked with a negative sign (-), and the position of the intercalating plug residue, Lys53, is marked with an asterisk (*). (B) Representative εA excision activity for Mag1, Mag2, and Mag2-Asp56Ser mutant. The image shows the electrophoretic separation of the 25mer εA -DNA substrate (S) from 12mer product (P) generated from alkaline cleavage of abasic sites produced by the glycosylase. Reaction times in hours are shown at the top. (C) Quantitation of εA activity data, shown as the average of three independent experiments.

Figure 5. Additional structural differences between Mag1 and Mag2 that likely impact DNA binding and base flipping

(A) Protein-DNA interactions around the abasic site. The Mag2 complex is colored blue (protein) and gold (DNA), and the Mag1 complex is cyan and silver. The phosphate backbone and main-chain protein atoms are traced with a cartoon. Only the damaged DNA strand is shown for clarity. The active site is at the top of the figure, and the outside of the protein (solvent) is at the bottom. (B) Solvent-accessible surfaces colored according to electrostatic potential (red, negative; blue, positive; −7 to +7 k_BT) calculated using DelPhi [52]. The DNA is colored as in Figure 2.